Medicines for relieving intestinal disorders

ABSTRACT

An agent for relieving intestinal disorders which contain live cells of a strain belonging to  Lactobacillus helveticus . This strain is preferably characterized by a resistance to a specific bile acid, adhesiveness to intestinal epithelial cells, etc. A method of controlling intestinal functions which involves the step of administering such a medicine to a subject with a need for the control of intestinal functions. When administered to humans or the like, these agents exert effects of elevating defecation frequency, etc., thereby efficaciously improving the intestinal environment.

FIELD OF ART

The present invention relates to an agent for relieving intestinaldisorders.

BACKGROUND ART

It is known that a variety of microorganisms live and form microorganicflora in an intestinal tract of humans and animals. Such microorganismsare classified into harmful microorganisms that give harmful effects tothe host, and beneficial microorganisms that give beneficial effects tothe host. These microorganisms keep symbiotic or antagonisticrelationship. The harmful microorganisms may be those which produceharmful products such as ammonia, hydrogen sulfide, and amines to placean excessive burden on liver function, and those which relates tocarcinogenesis. The harmful microorganisms may include genusClostridium.

Beneficial effects that are given to the host through improvement ofintestinal flora may include a variety of effects that will lead to goodintestinal environment which is necessary for the host to maintain ahealthy life, such as (1) amelioration of gastrointestinal conditionsuch as diarrhea or constipation, (2) prevention of cancer andimprovement of infection resistivity through activation of immunesystem, and (3) suppression of metabolic production of, e.g. harmfulenzymes, by the harmful microorganisms. Effective use of themicroorganic flora for the health of the host is the idea of so-calledprobiotics.

There are some proposal for definition of probiotics, such as “amicroorganism and a substance that give beneficial effect to a hostthrough control of intestinal flora” (Parker, R. B: An. Nutr. Health,29, 4-8 (1974)), “a live microorganism that gives beneficial effect to ahost by improvement of balance of intestinal florae” (Fullar, R.: J.Appl. Bacteriol., 66, 365-378 (1989)), “a strain of or mix-culturedbacteria that give a beneficial effect to a host by amelioration offactors relating to balance of intestinal florae in the host” (Havenaar,R. and Huis in't Veld, J. H. J.: A general view, In The Lactic AcidBacteria in Health and Disease/Wood, B. J. B. ed., pp.151-170 (1992)Elsevier, London), “live bacteria and substances promoting proliferationof such bacteria that give a beneficial effect to inherent flora of notonly animals but also plants and foods” (Fullar, R.: Probiotics: TheirDevelopment and Use, In Old Herborn University Seminar Monograph 8/vander Waaij, D., Heidt, P. J. and Rush, V. C. eds., pp.1˜8 (1995)Institute for Microbiology and Biochemistry, Herborn-Dill) (see TheJapanese Journal of Nutrition and Dietetics Vol.55 No.4 p167-177: 1997).In any case, a substance that is called probiotics has a function ofrelieving intestinal disorders.

It is reported that some of lactic acid bacteria have such probioticproperties. Specifically, it is known that some strains of Lactobacillusbulgaricus, Streptococcus thermophilus, Lactobacillus acidophilus,various bifidobacteria, Lactobacillus casei, and Lactobacillus gasserihave probiotic properties. More specifically, it is known thatLactobacillus plantarum 299DSM6596, Lactobacillus casei ssp. rhamnosus271DSM6595 (Japanese Patent No. 2742962 B), Lactobacillus acidophilusPN-RI-2-4 (JP-P-H5-292947 A), Lactobacillus acidophilus F-133 (JapanesePatent No.3052208 B), bifidobacteria BB536, and Lactobacillus caseiShirota strain have probiotic properties.

Particularly, as to the lactic acid bacteria of genus Lactobacillus, L.acidophilus, L. agilis, L. aviarius, L. amylovorus, L. brevis, L. casei,L. crispatus, L. delbrueckii subsp. bulgaricus, L. gallinarum, L.gasseri, L. johnsonii, L. murinus, L. hamsteri, L. intestinalis, L.plantarum, L. reuteri, L. ruminis, and L. salivarius are at presentrecognized as probiotics (probiotics A Critical Review: Gerald W.Tannock (1997) p47).

By the way, it is reported that (1) Lactobacillus helveticus has highproteolytic activity and thus has high peptide productivity, and (2) aLactobacillus helveticus-fermented milk has an hypotensive effect(Japanese Patent No.3028411 B). Therefore, it is known that anextracellular product obtained by fermenting a milk with Lactobacillushelveticus may be used as lactic acid fermented milk material withsuppressed lactic acid acidity (JP-P-H10-99018 A), stimulant forinterferon production (JP-P-S57-1237 A), and lipid metabolism improver(JP-P-H10-229841 A). However, it is not known that the live bacterialcells of Lactobacillus helveticus may be administered to hosts such ashumans for use as an agent for relieving intestinal disorders having aprobiotic function.

DISCLOSURE OF THE INVENTION

The object of the present invention is to provide an agent for relievingintestinal disorders that, when administered to a subject such as ahuman, leads to effects such as increased frequency of defecation and istherefore useful for improving the intraintestinal environment.

According to the present invention, there is provided an agent forrelieving intestinal disorders comprising live bacterial cells of astrain belonging to Lactobacillus helveticus.

According to the present invention, there is also provided a method forcontrolling intestinal function comprising the step of administering toa subject in need thereof an effective amount of the agent for relievingintestinal disorders.

EMBODIMENTS OF THE INVENTION

The agent for relieving intestinal disorders of the present inventioncontains live bacterial cells of a strain belonging to Lactobacillushelveticus.

The strain belonging to Lactobacillus helveticus is preferably resistantagainst bile acid. Specifically, the minimum growth inhibitoryconcentration of dry solid bile for the strain in a bile acidresistivity test may preferably be 0.8% or more, and more preferably1.3% or more.

The minimum growth inhibitory concentration of the dry solid bile is theminimum concentration of the dry solid bile at which concentration thebile can inhibit growth of the strain. Specifically, the minimum growthinhibitory concentration may be measured by preparing a liquid medium inwhich the strain belonging to Lactobacillus helveticus can grow (e.g.,MRS medium), adding a variety of concentrations (e.g., 0, 0.05, 0.1,0.3, 0.5, 1.0, 1.5 and 2.0%) of dry solid bile to the medium, culturingthe strain in each medium, measuring turbidity (e.g., at 650 nm) bye.g., absorptiometer after the lapse of certain periods of time (e.g.,0, 4, 8 and 12 hours) to determine specific growth rate (OD/time) at thelogarithmic growth phase, and calculating the concentration of the drysolid bile at which the specific growth rate becomes zero byextrapolation of the relationship between the dry solid bileconcentration and the specific growth rate. The dry solid bile mayspecifically be Bacto-oxgall (product name, manufactured by DifcoLaboratories).

The strain belonging to Lactobacillus helveticus preferably hasadhesiveness of 2.8×10⁶ cells/well or more, and preferably 4.5×10⁶cells/well or more to a monolayer culture of Caco-2 cells in anintestinal tract epithelium cell adhesiveness test in a cylindrical wellhaving a bottom surface area of 1.8 cm².

The adhesiveness to the intestinal epithelium cells may specifically betested, for example, in accordance with the Greene and Klaenhammer'smethod (Greene, J. D. and Klaenhammer, T. R. Applied and EnvironmentalMicrobiology 60, 4487-4494 (1994)). More specifically, for example, 1 mlof cultured liquid containing 1×10⁵ cells/ml of Caco-2 cells may bepoured in a cylindrical well having the bottom surface area of 1.8 cm²,and cultured until a monolayer is formed. Number of the cells of thestrain that can adhere to the monolayer of the cultured Caco-2 cells perwell may be measured. A strain exhibiting cell number that is not lessthan the aforementioned preferable number may preferably used.

The strain belonging to Lactobacillus helveticus may preferably be thosewhich exhibits fecal recovery in humans of 4.0×10⁷ cells or morefollowing administration of the strain at the dose of 1.0×10¹¹ cells.

The amount of the cells of the strain from the collected feces may bemeasured by the following method: the strain is made resistant againstan antibiotic such as rifampicin in accordance with an ordinary method;the resistant bacteria are administered to a human subject; the feces ofthe subject is collected after the administration; and the number of thebacterial cells in the feces is counted utilizing the antibioticresistivity. The amount of the strain in the collected feces may also bemeasured by detecting genes derived from the strain in the feces inaccordance with an ordinary method.

As the strain belonging to Lactobacillus helveticus, a variety ofstrains such as Lactobacillus helveticus CP53 strain (InternationalPatent Organism Depositary of National Institute of Advanced IndustrialScience and Technology, deposition number FERM BP-5770) maybe used.Lactobacillus helveticus CP53 strain is particularly preferable.

Lactobacillus helveticus CP53 strain has been deposited at NationalInstitute of Bioscience and Human-Technology Agency of IndustrialScience and Technology (which is at present called International PatentOrganism Depositary of National Institute of Advanced Industrial Scienceand Technology) on Jan. 11, 1996 as the deposition number FERM BP-5770,and is now publicly available.

The formulation of the present agent for relieving intestinal disordersis not particularly limited and may be any of solids such as powders,granules and tablets; and fluids such as pastes, gels and liquids.

The amount of the live bacterial cells of the strain in the agent forrelieving intestinal disorders of the present invention is notparticularly limited. However, the amount may preferably be those whichenables ingestion of 1.0×10¹¹ or more of the live bacterial cells at oneadministration. Specifically, the content of the live bacteria may be1.0×10⁶ cells/g to 1.0×10¹¹ cells/g, and preferably 1.0×10⁸ cells/g to1.0×10¹¹ cells/g.

Although the method for producing the agent for relieving intestinaldisorders may not be limited, the agent may be produced by fermentingmilk with a strain belonging to Lactobacillus helveticus, to obtain livebacterial cells of the strain belonging to Lactobacillus helveticus. Themilk may be animal milk such as cow's milk, horse milk, goat milk andsheep milk; and vegetable milk such as soybean milk. The fermentationtemperature may be 20° C. to 50° C., and preferably 30° C. to 45° C. Thefermentation time may preferably be 3 to 48 hours, and preferably 6 to24 hours. The fermented product itself obtained by fermentation of themilk with the strain belonging to Lactobacillus helveticus may beutilized as the agent for relieving intestinal disorders of the presentinvention. The fermented product may further be admixed with othermaterials such as other additives and food materials, and optionallyprocessed to be in a form such as powders, granules and tablets, for useas the agent for relieving intestinal disorders of the presentinvention. Alternatively, the live bacterial cells of the strainbelonging to Lactobacillus helveticus cultured in accordance with theaforementioned fermentation or other methods may be collected from themedium, and the collected bacteria itself may be utilized as the agentfor relieving intestinal disorders of the present invention. Thecollected bacteria may further be admixed with other materials such asother additives and food materials, and optionally processed to be in aform such as powders, granules and tablets, for use as the agent forrelieving intestinal disorders of the present invention.

The subject to which the agent for relieving intestinal disorders of thepresent invention is administered may be animals such as mammalsincluding humans, without limitation.

The dose of the agent for relieving intestinal disorders of the presentinvention may be 1.0×10¹¹ cells in terms of number of the live bacterialcells per one day in the case of human. The agent may be administered ata time or a plurality of times.

When the agent for relieving intestinal disorders of the presentinvention is administered to an animal including human, it may result inrelief of intestinal disorders represented by increased frequency ofdefecation and amount of feces.

The agent for relieving intestinal disorders of the present inventiongives an effect such as the increased frequency of defecation whenadministered to an animal such as a human, and is therefore useful forimproving the intraintestinal environment.

EXAMPLES OF THE INVENTION

The present invention will be explained more in detail with reference tothe Examples. However, the present invention is not limited thereto.

In the following Examples, adhesiveness to Caco-2 cells was measured asfollows:

Test of Adhesiveness to Caco-2 Cells

Adhesiveness to Caco-2 cells was measured in accordance with Greene andKlaenhammer's method. Caco-2 cells purchased from American Type CultureCollection (HTB-37) in MEM medium (Gibco BRL) containing 5% calf serumalbumin (Gibco Bethesda Reserarch Laboratory) that had been inactivatedby heating at 55° C. for 30 minutes were cultured at 37° C. in 5% CO₂incubator. 1 ml of the cultured liquid containing 1×10⁵ cells/ml wastransferred to a cultivation plate having 24 wells (manufactured bySumitomo Bakelite Co., Ltd., trade name MS-80240). The medium wasexchanged every 48 hours until the cells form a monolayer. 0.1 ml of 20%glutaraldehyde (Wako Pure Chemical Industries, Ltd.) was added forfixing the cells to the culturing plate. The plate was left stand for 30minutes and washed two times with PBS. 0.5 ml (1×10⁹ cells) of a liquidcontaining cells to be tested that had been isotope-labeled were added.After culturing at 37° C. for one hour, free cells were removed bywashing the plate three times. 200 μl of 1% SDS, 1N NaOH was added andleft stand for 30 minutes. The mixture of the Caco-2 cell and thebacterial cells were recovered from the wells, and suspended in ascintillator cocktail (Scintisol EX-H, Wako Pure Chemical Industries,Ltd.) and radio activity thereof was measured with a liquidscintillation counter LSC-900 (Aloka Co., Ltd.). The number of the cellsadhered was calculated on the basis of specific radioactivity of thecells that had previously been counted. The measurement was performedtwice and average was taken as the result.

Example 1

Lactobacillus Helveticus CP53 Strain

Lactobacillus helveticus CP 53 strain is known to have therein a plasmidDNA, pCP53 (Yamamoto, N. and Takano, T.: Biosci. Biotech. Biochem., 60(12), 2069-2070 (1996)). Lactobacillus helveticus CP53 strain wascultured in a variety of MRS mediums each containing differentconcentration of solid bile powders (Bacto-oxgall (Difco)) for measuringminimum growth inhibitory concentration. The minimum growth inhibitoryconcentration was found to be 1.31%. Adhesiveness to Caco-2 cells wasalso measured and confirmed to be 4.5×10⁶ cells/well or more.

Conferring Rifampicin Resistance

For recovery from feces in the ingestion test, resistance against theantibiotic rifampicin was conferred to CP53 strain in accordance with anordinary method. The CP53 strain was spread on a MRS plate mediumcontaining 100 μg/ml of rifampicin. The rifampicin-resistant strain thatgrew on this plate medium was selected as CP53-R strain.

Properties of CP53-R Strain

CP53-R strain was subcultured seven times in MRS medium that did notcontain rifampicin. The subcultured strain was cultured in a variety ofMRS mediums each containing different concentration of solid bilepowders for measuring the minimum growth inhibitory concentration, whichwas found to be 0.86%. Adhesiveness to Caco-2 cells was also measuredand confirmed to be 2.8×10⁶cells/well or more. Further, this subculturedstrain was confirmed to contain the plasmid pCP53.

Ingestion-Recovery Test on Volunteers

CP53-R strain cultured in MRS medium (Difco) was collected bycentrifugation, washed twice with PBS, and suspended in cow's milk at aconcentration of 1×10¹¹ cells/100 ml. The milk suspension containingCP53-R strain was employed as an ingestion sample for theingestion-recovery test. Seven healthy subjects at the age of 25 to 41drunk 100 ml of the ingestion sample at once. All of feces defecatedfrom the subjects within 5 days from the ingestion were collected.Frequency of defecation, and amount and hardness of feces from eachsubject after administration of the ingestion sample were recorded. Theresults are shown in Table 1. The collected feces was immediately frozenand stored at 4° C., and the following analyses were performed on thenext day.

Survival of L. Helveticus

Utilizing the MRS plate medium containing rifampicin, number of livebacterial cells having rifampicin resistance in the feces samples wascounted. The number of bacterial cells contained in all of the collectedfeces was calculated. As a result, average number of recovered bacterialcells per person was 1.2×10⁸ cells. It was confirmed that all of thebacteria survived on the MRS plate medium containing rifampicin had theplasmid pCP53, i.e., they were CP53-R strain.

Intraintestinal Growth of L. Helveticus

CP53 strain cells were washed twice with PBS, and subjected to alkalinebacteriolysis with 10 mg/ml lysozyme (Seikagaku Corporation) and 1 mg/mlof mutanolysin (Seikagaku Corporation), to prepare pCP53 plasmid DNA.The DNA was then purified by high speed centrifugation with CsCl densitygradient, and radioisotope-labeled with Random Primer DNA Labeling Kitver 2.0 (Takara Bio Inc.) and [α-³²P] dCTP (222 TBq/mmol, New LifeProducts Inc.), to obtain a ³²P radio-labeled pCP53 plasmid DNA.

In a centrifugation tube of 15 ml volume, 5 ml of 30% sucrose solution,2 ml of 20% sucrose solution and 2 ml of 10% sucrose solution werecalmly piled up, to obtain a step density gradient sucrose solution. Thefeces sample collected from the subjects was diluted 20 times (w/w) andsuspended in a distilled water, and 0.5 ml of the suspension was laid onthe step gradient sucrose solution. The laid liquids were centrifugedfor five minutes at 1500×g. 10% and 20% sucrose fractions werecollected, and washed twice with sterilized water. A DNA fraction wasobtained by the Leenhouts' method (Leenhouts, K. J., Kok, J., andVenema, G. (1990) Appl. Environ. Microbiol., 56, 2726-2735). Theobtained DNA fraction was dissolved in 10 mM Tris Cl pH 8.0, 1 mM EDTA.10 and 100 times dilutions of the solution with the same buffer werealso prepared. These DNA fractions were heated for three minutes and 10μl each thereof was dot-blotted on a nylon membrane (Hybond-N⁺, AmershamPharmacia Biotech). The blotted nylon membrane was washed with distilledwater and dried, to fix DNA.

To the fixed DNA on the membrane, the aforementioned radio-labeled pCP53plasmid DNA was hybridized in accordance with Maniatis' method(Maniatis, T., Frish, E. F. and Sambrook, J. (1987) Molecular Cloning; alaboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor).Upon hybridization, formaldehyde and chromosomal DNA derived from avariety of bacteria in separately prepared feces that did not containCP53 strain were added thereto at 50% (w/w) and 10 μg/ml, respectively,for avoiding non-specific hybridization.

For quantification of pCP53 plasmid, the membrane after hybridizationwas subjected to autoradiography, and each sample dot portion (0.5×0.5mm) was cut off. Radio activity of the taken dot portions were measuredwith a liquid scintillation counter LSC-900 (Aloka Co., Ltd.).

Separately, for obtaining a calibration curve, 8.5×10³ to 2.0×10⁵ cellsof CP53 strain was mixed with feces to prepare feces samples, which weresubjected to the aforementioned DNA fraction preparation, dot-blottingand reaction with radio-labeled pCP53plasmid DNA. The bound radioactivity was in linear proportion to number of CP53 strain cells thathad been mixed with feces in a range of 8.5×10³ to 2.0×10⁵ cells/g(weight of feces), and a calibration line was thus obtained. With thiscalibration line, amount of CP53 strain cells in the feces samples fromthe ingestion-recovery test subjects was determined in a range of 1×10⁴to 2.0×10⁵ cells/g (weight of feces)

As a result, CP53 strain cells were found in all of the feces samplescollected from the seven subjects. Number of total CP53 strain cells inall of feces samples from each subject was 4.0×10⁹, 1.0×10⁹, 3.0×10⁹,1.0×10⁹, 1.0×10⁹, 4.0×10⁷ and 4.0×10⁸ cells, respectively. In eachsubject, CP 53 strain was found in all of the feces samples of thefirst, second and third defecation after taking the ingestion sample.The amounts of CP53 strain cells in the feces samples of the third andsubsequent defecation were greater than that of the first and seconddefecation. This fact suggests that CP53 strain can grow and proliferatein the intestinal tract even it can hardly grow in feces.

Feces from subjects who had not ingested CP53-R live bacteria was alsotaken as feces sample, and DNA fractions were prepared therefrom in thesame way as the above. These DNA fractions and purified pCP53 plasmidDNA were dot-blotted and reacted with the radio-labeled pCP53 plasmidDNA in the same way. As a result, the radio-labeled pCP53 plasmid DNAstrongly reacted with purified pCP53 plasmid, but did not react at allwith the DNA fractions derived from the feces from the subjects who hadnot ingested CP53-R live bacteria.

Comparative Example 1

Example 1 was followed except that the ingestion sample washeat-sterilized before administration to the subjects. Frequency ofdefecation, and amount and hardness of the feces of each subject wererecorded. The results are shown in Table 1.

TABLE 1 Example 1 Comp. Example 1 Frequency of  5.6 ± 1.9* 4.6 ± 1.6defecation (times/5 days) Amount of feces 140 ± 57  130 ± 48  (ml/day)Hardness (as to the  1.9 ± 0.38  1.9 ± 0.69 first feces after intake ofthe ingestion sample)^(a)) *Significant compared to Comparative Example1 (p < 0.1, Wilcoxon's signed rank test) ^(a))Evaluated in threedegrees: 1; hard, 2; normal, 3; soft. Average was calculated.

Formulation Example 1

1.485 kg of skim milk powders (manufactured by Yotsuba Inc.) weredissolved in 8.415 kg of purified water. The solution washeat-sterilized at 95° C. for 30 minutes and then quickly cooled down to32° C. To the recombined and sterilized skim milk was aseptically added0.1 kg of bulk starter of Lactobacillus helveticus CP 53 strain. Themixture was thoroughly stirred and fermented at 32° C. for 16 to 20hours. The fermentation was terminated when the acidity of the fermentedmilk (weight percentage of lactic acid) reached in a range of 1.5 to2.1. After finishing the fermentation, the fermented milk was quicklycooled down to 10° C. or less to obtain 10 kg of fermented milkmaterial. At that time, number of live CP53 strain cells per 1 g reachedat least 1.5 billion.

0.8 kg of granulated sugar powders and 0.03 kg of pectin YM115-H powders(manufactured by Copenhagen Pectin) were mixed and dissolved in 4.17 kgof purified water warmed at 70° C. The solution was heat-sterilized at95° C. for several seconds and then quickly cooled down to 10° C. orless to obtain 5 kg of sterilized sugar-stabilizer solution.

5 kg of the fermented milk material was aseptically added to 5 kg of thesterilized sugar-stabilizer solution and the mixture was asepticallyhomogenized with a laboratory homogenizer (type: 15M-8BA, manufacturedby APV Gaulin) at the homogenizing pressure of 15 MPa and processingflow rate of 2.5 L/min, to obtain 10 kg of dairy lactic acid bacteriabeverage bulk. The dairy lactic acid bacteria beverage bulk wasaseptically poured into polystyrene bottles of 200 g size. The bottleswere heat-sealed with polyethylene-aluminum laminated films, to obtaindairy lactic acid bacteria beverage products having quality preservationperiod of 14 days in refrigerator (10° C. or less).

The obtained product contained at least 750 million CP53 strain cellsper 1 g. When preserved at 10° C. or less, the product was able tomaintain 500 million live bacterial cells per 1 g even at 14 days afterproduction. Therefore, drinking 200 g of the product results iningestion of 100 billion or more of live CP53 bacterial cells.

Formulation Example 2

0.180 kg of skimmed soybean milk powders (trade name “Soyafit 2000”manufactured by Fuji Oil Co., Ltd.) was dissolved in 1.738 g of purifiedwater, which was then admixed with 0.080 kg of lactose and 0.002 kg ofyeast (trade name “Ebios P2G” manufactured by Asahi Breweries, Ltd.) andstirred. The mixture was sterilized at 95° C. for several seconds andthen quickly cooled down to 25° C. To the recombined and sterilized skimsoybean milk thus obtained was aseptically added 0.060 kg of bulkstarter of Lactobacillus helveticus CP53 strain. The mixture wasthoroughly stirred and fermented at 37° C. for 24 to 48 hours withstirring at 100 rpm. The fermentation was terminated when the acidity ofthe fermented milk reached in a range of 1.5 to 2.1. After finishing thefermentation, the fermented soyabean milk was quickly cooled down to 10°C. or less to obtain 2 kg of fermented soyabean milk material. At thattime, number of live CP53 bacterial cells per 1 g reached at least 2billion.

0.44 kg of granulated sugar powders and 0.015 kg of pectin YM115-Hpowders (manufactured by Copenhagen Pectin) were mixed and dissolved in3.56 kg of purified water warmed at 70° C. The solution washeat-sterilized at 95° C. for several seconds and then quickly cooleddown to 10° C. or less to obtain 4 kg of sterilized sugar-stabilizersolution.

2 kg of the fermented soyabean milk material was aseptically added to 4kg of the sterilized sugar-stabilizer solution and the mixture wasaseptically homogenized with a laboratory homogenizer (type: 15M-8BA,manufactured by APV Gaulin) at the homogenizing pressure of 15 MPa andprocessing flow rate of 2.5 L/min, to obtain 6 kg of soyabean lacticacid bacteria beverage bulk. The soyabean lactic acid bacteria beveragebulk was aseptically poured into polystyrene bottles of 200 g size. Thebottles were heat-sealed with polyethylene-aluminum laminated films, toobtain soyabean lactic acid bacteria beverage products having qualitypreservation period of 14 days in refrigerator (10° C. or less).

The obtained product contained at least 660 million CP53 strain cellsper 1 g. When preserved at 10° C. or less, the product was able tomaintain 500 million live bacterial cells per 1 g even at 14 days afterproduction. Therefore, drinking 200 g of the product results iningestion of 100 billion or more of live CP53 bacterial cells.

1. A method for controlling intestinal function, wherein the control isincreased frequency of defecation and amount of feces, comprising thestep of administering to a subject in need thereof an effective amountof an agent comprising live bacterial cells of a strain belonging toLactobacillus helveticus, wherein the minimum growth inhibitoryconcentration of dry solid bile for said strain is at least 0.8% asdetermined by a bile acid resistivity test for said strain, wherein saidstrain has an adhesiveness of 2.8×10⁶ cells/well or more to a monolayerculture of Caco-2 cells in a cylindrical well having a bottom surfacearea of 1.8 cm² in an intestinal tract epithelium cell adhesion test,and wherein said strain has a fecal recovery in humans of 4.0×10⁷ cellsor more following administration of the strain at a dose of 1.0×10¹¹cells.
 2. The method of claim 1, wherein said strain is Lactobacillushelveticus CP53 strain (International Patent Organism Depositary ofNational Institute of Advanced Industrial Science and Technology,deposition number FERM BP-5770).
 3. The method of claim 1, wherein saidagent is administered at a dose of 1.0×10¹¹ cells in terms of number ofthe live bacterial cells per one day.